Summary of the November 2013 Issue of BioTechniques
For the November 2013 issue of BioTechniques, in honor of the 30th anniversary of the publication of BioTechniques, we will be presenting a very special ‘retro’ issue of the journal. While this special ‘retro’ issue will contain all new news features and research articles, the articles will be presented in a format and style reminiscent of BioTechniques articles from the early 1990’s. This issue will also contain a look at the evolution of the journal cover art during the past three decades as well as a special news feature exploring the development of key technologies and the role BioTechniques has played in their communication to the greater scientific community.
This November issue will feature six new research articles focusing on topics including, the use of extended autoclaving to remove contaminating waste DNA, a new approach to microsatellite discovery using the PacBio sequencing system, a method for eliminating rRNA from RNA sequencing libraries, a technique for creating isogenic inducible cell lines, and an article detailing how replication timing can influence array-CGH copy number determination.
A large number of methods are available to deplete ribosomal RNA reads from high throughput RNA sequencing experiments. Such methods are critical for sequencing Drosophila small RNAs between 20 and 30 nucleotides because size selection is not typically sufficient to exclude the highly abundant class of 30 nt 2S rRNA. In a report slated for the November issue, researchers from the University of Kansas demonstrate that pre-annealing terminator oligos complimentary to Drosophila 2S rRNA prior to adapter ligation and reverse transcription efficiently depletes 2S rRNA sequences from the sequencing reaction enhancing sequencing library preparation.
In an article that will appear in the November issue, scientist from Germany consider whether there is a direct biological impact from cell cycle dynamics on the output of DNA copy number detection by array-CGH. The proportion of actively replicating cells in highly proliferating cell cultures or tissues is responsible for the non-equimolar DNA content. This is directly visualized in array-CGH data analysis as copy number waves, frequently discussed by the array-CGH community rather as a technical error, and shows a remarkable similarity to replication timing profiles of respective organisms and tissues of origin.
Manipulating gene expression in mammalian cell lines is perhaps one of the most widely used methods to study gene functions. Tetracycline- and doxycycline-inducible systems are sensitive, reproducible, relatively inexpensive, and proven to work well both in cell lines and mouse models. To obtain homogeneous transgene expression or uniform knockdown by short hairpin RNA, however, requires time-consuming and labor-intensive single-cell cloning to derive stable cell lines, a procedure which hinders the Tet-inducible cells to be widely adopted by researchers. A team from Johns Hopkins University describes the establishment of a novel system (the XT-cell method) in establishing isogenic inducible cell lines using founder reporter lines and recombinase-mediated cassette exchange in the November issue of BioTechniques. Using this XT-cell method, the isogenic stable Tet-inducible cell lines can be efficiently created with much less effort and time as compared to conventional methods.
High-throughput microscopy is an effective tool to rapidly collect large-scale datasets. However, high throughput comes at the cost of low spatial resolution. In the November issue, a team from the University of Heidelberg introduces correlative light microscopy, a combination of fast automated widefield imaging, confocal microscopy and super-resolution microscopy. The authors demonstrate the potential of the approach for scalable experiments and as a robust approach for selecting cells of interest on a wide-field screening microscope at low resolution and subsequently relocalizing those cells with micrometer precision for confocal and super-resolution imaging.
Carryover and false-positive amplification of undesired DNA sequences have been serious problems in experiments and diagnoses using PCR. In a report in the November, a team of researchers detail how although autoclaving at 121°C for 20 min did not sufficiently remove template activity of DNA and was found to be a possible source of laboratory contamination, the template activity of DNA could be removed by autoclaving at 121°C for 100 min.
Microsatellite sequences are important markers for population genetic studies. However, the development of adequate microsatellite primers has been cumbersome in the past. With the advent of next generation sequencing technologies, marker identification in genomes of non-model species has been greatly simplified. In another report in the November issue, researcher from Germany describe microsatellite discovery on a Pacific Biosciences RS single molecule sequencer. Combined with a straight forward amplification-free library preparation, PacBio sequencing is an economically viable alternative for microsatellite discovery and subsequent PCR primer design.
In addition to the research articles in November, the Tech News article for this month focuses on the development and evolution of PCR, sequencing and cell imaging through interviews with key figures and thought leaders in these areas of methods development.
Keywords: PCR, next-generation sequencing, RNA seq, microsatellites, DNA contamination, confocal microscopy, light microscopy, super-resolution imaging, high-throughput imaging and screening, PCR primer design, array-CGH, cell cycle timing, inducible cell lines, shRNA, inducible expression vectors